Non-invasive Imaging of Leukocyte Homing and Migration in vivo
Summary
Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.
Abstract
Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.
Protocol
Discussion
In this video protocol we demonstrate the procedures for non-invasive 2P imaging of leukocyte recruitment in response to inflammation.
The footpad is a classic physiological site for studying inflammation such as allergy, infection and autoimmune disease 4-7. 2P imaging provides a detailed picture of distinct steps in leukocyte trafficking pathways, from rolling and adhesion in the blood vessels to chemotaxis to sites of effector function. We have found that the leukocyte recrui…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIH grant R01-3155-53502 and Washington University-Pfizer Biomedical Research Agreement.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Isoflurane, USP | Butler Animal Health Supply | 029405 | ||
| 655nm non-targeted quantum dots | Invitrogen | Q21021MP | ||
| Tetramathylrhodamine conjugated E. coli BioParticles |
Invitrogen | E2862 | ||
| Listeria monocytogenes (Lm) | Reference 2 | |||
| Quick gel | Duro | |||
| Puralube Vet Ointment | Pharmaderm Animal Health | |||
| Insulin syringes | BD | 08290-3284-38 | ||
| PBS | Thermo Scirntific | SH30256.02 |
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